We are proposing to study the active site, examine possible accessory catalytic activity, and characterize proteases which cause degradation of the enzyme, terminal deoxynucleotidyl transferase (TdT), which is purified to homogeneity from calf thymus gland. For the first objective, we will synthesize several fluorescent nucleotides which are substrates or inhibitors of TdT. These will be used to make steady-state and time-resolved fluorescence measurements to determine binding constants and approximate the active site size restriction and exclusion. The information which we obtain about the catalytic domain may aid in development of active site directed inhibitors which may be useful as chemotherapeutic agents for certain leukemias in which high levels of TdT are expressed. For the second objective we will determine whether the TdT protein has catalytic function unrelated to non-template directed polymerization. We have evidence that an endonuclease which produces double strand breaks in superhelical DNA may be associated with the TdT molecule. Since current theories of the function of TdT are related to recombinational events which could generate diversity in DNA sequences in cells of the immune system, suggestion of an associated endonuclease activity could offer a mechanism for how such events are initiated in TdT (+) cells. The biochemical characteristics and substrate specificity of this endonuclease will be studied, and association with TdT protein will be ascertained. For the third objective we will isolate and characterize proteolytic enzymes in calf thymus which cleave the TdT molecule from a high molecular weight single polypeptide to a structure comprised of two non-identical subunits. Throughout this proteolysis, a two-fold reduction in mass occurs although TdT activity remains conserved. We will use radiolabeleld high molecular weight TdT as a substrate to identify these proteolytic enzymes, compare TdT degradation profiles with those of known proteolytic enzymes, and determine which protease inhibitors can be used during purification of TdT so that sufficient high molecular weight TdT can be isolated for biochemical characterization.